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Scientists know that carrying the R47H variant of the TREM2 gene triples a person’s chances of developing Alzheimer’s disease, but they don’t yet know how the mutation affects the function of this microglial receptor in vivo. In the January 10 Journal of Experimental Medicine, Marco Colonna and colleagues at Washington University in St. Louis report that activated microglia do not surround plaques in mice expressing the mutated version of the human protein the way they do in AD mice with the common TREM2 variant. Moreover, they found that, normally, soluble TREM2 released from cell membranes latches onto Aβ plaques and nearby neurons, but the R47H variant significantly loses this binding. Overall, their model suggests that R47H impairs TREM2 function and supports the idea that normal TREM2 plays a protective role in disease.

Human TREM2 expressed in AD mouse models.

The R47H AD risk variant activates microglia only weakly.

Soluble TREM2 binds neurons, plaques. Soluble R47H: Not so much.

“This is a very important paper,” wrote Christian Haass, Ludwig-Maximilians University, Munich, to Alzforum (see comment below). “It provides clear evidence that the most relevant AD-associated variant of TREM2, R47H, causes a partial loss of function.”

TREM2 has been proposed to activate microglia, especially those around plaques, and soluble TREM2 cleaved from the membrane reportedly enhances microglial survival and stokes inflammation (Apr 2017 conference news; Zhong et al., 2017). Previous studies from Colonna and others suggested R47H TREM2 binds ligands more weakly in vitro than does the wild-type receptor, while AD patients who carry the R47H variant have fewer plaque-associated microglia than other AD patients (Wang et al., 2015; Yuan et al., 2016). Together, these results suggest a loss of function for R47H, but scientists have yet to confirm this in an in vivo model.

To do that, first authors Wilbur Song and Satoru Joshita knocked out TREM2 in 5XFAD mice and introduced instead the human version of the gene. They engineered mice to express either the common variant (CV) of TREM2 or the R47H version on a bacterial artificial chromosome (BAC) that included regulatory sequences for TREM2. The researchers then compared the brains of these BAC lines at 8.5 months.

As in people, the full-length TREM2 protein appeared exclusively in microglia, with control and R47H versions expressed at similar levels. However, because no known reagent labels both human and mouse TREM2, the scientists were unable to compare BAC gene expression with that of endogenous TREM2 in wild-type mice.

Nevertheless, staining hippocampal and cortical slices of the BAC mice revealed that slices from the CV variant had more activated microglia near plaques than did those from the R47H variant. Microglia from CV mice also expressed higher levels of transcriptional markers of activation, including Spp1 and Gpnmb. However, in both TREM2 strains, soluble Aβ levels and plaque area in the cortex and hippocampus equaled those of 5XFAD-TREM2 knockouts. This suggests TREM2 has no effect on Aβ plaques before 8.5 months, at least in these animals, which begin to aggressively deposit Aβ by 1.5 months of age.

To the researchers’ surprise, the shed, soluble, i.e. extracellular domain of human TREM2 turned up on cell bodies of seemingly healthy neurons and in Aβ plaques in the cortex and hippocampus. The R47H mutation seemed to impede this binding, because the authors detected five times more bound sTREM2 in CV than in R47H mice, despite equal expression.

“That was really an unexpected finding,” Colonna told Alzforum. “We anticipated some soluble TREM2, but we didn’t expect it to bind so closely to cells and to plaques.” Colonna said it is unclear what the soluble protein binds to, or how that interaction affects neurons and plaques. He plans to find out.

What might this have to do with ectodomain shedding? Previously, Haass reported that ADAM proteases cleave R47H TREM2 less efficiently than normal in HEK293 and microglial cell lines (Kleinberger et al., 2014; Jul 2014 webinar). Now, Song and colleagues report that ADAM17 was equally effective at shedding both in macrophage-like cultures. Haass thinks overexpression of TREM2 in these cells might affect shedding; Colonna thinks both wild-type and R47H might be shed equally but the former binds better to plaques and neurons.

Regardless, Haass considers this binding crucial. “This paper shows nicely that we should not overlook the function of shed sTREM2,” he wrote. “Now we need to learn what sTREM2 is doing on the surface of neurons and within plaques. And maybe this will finally refocus AD researchers on the function of soluble APP, which is shed by the same proteases as TREM2, namely ADAM 10 and 17.” Jochen Walter at the University of Bonn, Germany, agreed. “It will be interesting to assess whether this binding of soluble TREM2 has a functional effect,” Walter wrote to Alzforum.

Taken together, the data suggest a partial loss of function of R47H. This agrees with a paper from Haass’ group on the T66M TREM2 mutation associated with frontotemporal dementia, which also reduced microglial clustering and activation in vivo (May 2017 news on Kleinberger et al., 2017). It will be important to observe these mice at later time points to see if TREM2 has the same positive impact, said Colonna, who made his mouse models available through his lab.

“Overall, the hTREM2 mice produced by the Colonna lab will be valuable as animal models to understand the biology of AD-associated variants, test potential TREM2-targeted therapies, and investigate the regulation and possible function, of sTREM2,” wrote Jason Ulrich, Washington University School of Medicine in St. Louis, to Alzforum (see full comment below). “It will also be interesting to compare the effect of variants in human TREM2 to the numerous TREM2 variant knock-in mice that are being generated,” Ulrich added.—Gwyneth Dickey Zakaib

Comments

The data from the Colonna group’s human TREM2 BAC transgenic mice provide another important layer of insight into the role of TREM2 and TREM2 variants in Alzheimer’s disease. The decreased plaque-associated microgliosis in both 5xFAD-TREM2 KO and 5xFAD-R47H mice compared to the 5xFAD-CV is consistent with the hypothesis that the R47H variant impairs TREM2 function leading to a reduction in the microglial response to amyloid pathology. These data are also in agreement with observed decreases in plaque-associated microgliosis and activation in postmortem R47H variant AD tissue (Krasemann et al., 2017; Yuan et al., 2016). The observation that the R47H and CV exhibited similar levels of surface expression is also in agreement with previous studies that characterized the effect of TREM2 variants on the protein’s trafficking and maturation (Kleinberger et al., 2014).

Perhaps the most intriguing set of data in the paper relates to the detection of TREM2 extracellular domain on neuronal cells and amyloid plaques. Analysis of sTREM2 levels in the CSF of AD cohorts would suggest that sTREM2 elevations occur subsequent to amyloid deposition and seem to best correlate with CSF tau levels rather than Aβ (Piccio et al., 2016; Suárez-Calvet et al., 2016; Suárez‐Calvet et al., 2016). Does the apparent increase in sTREM2 staining in BAC transgenic mice, particularly that fraction co-localizing with neurons, relate to the elevations in CSF sTREM2 that correlate with markers of synaptic damage? Even more provocatively, does sTREM2 have a physiological function in regard to neuronal health? It is interesting that sTREM2 was not detected on dystrophic neurites, but rather neuronal soma. Conceivably the axons of sTREM2-positive neurons could exhibit dystrophy, but the question remains as to why the sTREM2 would seem to accumulate at the cell body.

Overall the hTREM2 mice produced by the Colonna lab will be very valuable as an animal model to understand the biology of AD-associated variants, test potential TREM2-targeted therapies, and investigate the regulation, and possible function, of sTREM2. It will also be interesting to compare the effect of variants in human TREM2 to the numerous TREM2 variant knock-in mice (T66M, Y38C, R47H, etc.) that are being generated.

This is a very important paper. Colonna and colleagues provide clear evidence that the most relevant AD-associated variant (TREM2 p.R47H) causes a partial loss of function. The authors show this by an elegant approach. They rescue TREM2 KO phenotypes by transgenic overexpression of human wt TREM2 or the AD-associated variant p.R47H. Whereas wt TREM2 rescued microgliosis and microglia activation in 5xFAD mice, the mutant showed reduced rescuing activity. Thus the picture becomes more and more clear: TREM2 activity must have a protective function and microglial activation may be beneficial, at least initially. This is fully in line with our recent findings on the p.T66M mutation (Kleinberger et al., 2017), which strongly reduces microglial clustering and activation in vivo. Of note, these findings have important implications for therapeutic strategies aiming to modulate TREM2-dependent microglial activity.

I also think that this paper shows nicely that we should not overlook the function of shed sTREM2. Colonna and his colleagues show that it accumulates in plaques and neurons. Increased shedding of sTREM2 during plaque accumulation also fits nicely with our CSF data from human patients (Suarez-Calvet et al., 2016; Suarez-Calvet et al., 2016), which showed that sTREM2 increases early during disease development. Now we need to learn what sTREM2 is doing on the surface of neurons and within plaques. And maybe this will finally refocus AD researchers on the function of soluble APP, which is shed by the same proteases as TREM2, namely ADAM 10 and 17 (Kleinberger et al., 2014; Schlepckow et al., 2017; Thornton et al., 2017).

This is the second paper that back-translates human TREM2 mutations into mice. The first one was the T66M mutation by the Haass group (Kleinberger et al., 2017).

Besides offering important basic insights, this pivotal study adds supporting evidence on the directionality for potential TREM2-related therapeutic interventions in favor of TREM2 enhancing therapies. It seems that both membrane bound WT-TREM2 as well as shed WT-sTREM2 contribute to the positive modulatory role in the 5xFAD amyloidosis model when the R47H and WT TREM2 variants are compared. This picture gains importance by the fact that the authors introduced human TREM2 WT and R47H variants into a TREM2-deficient mouse background followed by a cross-breeding to the 5xFAD amyloidosis model.

Given the role of TREM2 on microglia activation, as established in the paper, it might emerge as a target that could be effective in AD at later stages, although additional work is needed to substantiate this idea.

Further, the binding of sTREM2 (WT, not for the R47H variant) on neurons is new and unexpected and will certainly stimulate efforts to identify the binding site and reveal its function (e.g., conferring neuroprotection?).

The new study by Song et al. describes the generation and analysis of transgenic mice expressing the human common variant (CV) of TREM2 or the R47H variant that has been associated with Alzheimer’s disease (AD).

This study is interesting not only because it is the first demonstration of transgenic mice expressing human R47H TREM2, but also reveals functional effects of this variant in brain resident microglia in vivo, despite limited effects on amyloid deposition.

The Colonna group generated BAC transgenic mice expressing either the human common variant (CV) or the AD-associated R47H variant of TREM2 on a TREM2 knockout background. Detailed expression analysis of two lines showed overall similar expression of CV or R47H mutant TREM2 protein at the surface of isolated macrophages, although mRNA levels were about 20 percent higher and gene copy number was fivefold higher in the brains of CV TREM2 transgenic mice as compared to R47H TREM2 transgenic mice.

Interestingly, bone marrow-derived macrophages from R47H TREM2 mice showed decreased viability upon CSF1 withdrawal from the culture media as compared to that of CV TREM2 mice, suggesting decreased activity of the R47H TREM2 variant in the promotion of cell survival.

To assess the role of human CV and R47H TREM2 during amyloid deposition, these mice were crossed with a transgenic (5XFAD) amyloidosis mouse model. The mice expressing human CV TREM2 had higher levels of microgliosis (higher number of microglia) around plaques as compared to R47H TREM2 transgenic mice. However, the plaque load was comparable in both CV and R47H TREM2 transgenic mice. The higher microgliosis around plagues might be related to increased viability and higher activation of microglia expressing CV TREM2 than that expressing the R47H variant. Indeed, authors show profound upregulation of several transcripts related to activation of microglia, including Cst/, Spp1, and Gpnmb in brains of CV TREM2 transgenic mice as compared to that expressing the R47H variant.

Another interesting finding in this study is that soluble TREM2 appears to bind to plaques and to neurons. Here, binding of soluble CV TREM2 was higher as compared to R47H TREM2. It remains to be determined to which structures soluble TREM2 binds on neurons and plaques. As discussed in the paper, phospholipids and Aβ are potential candidates. The binding of soluble TREM2 to neurons might also underlie earlier findings of TREM2 reactivity on neurons in APP transgenic mice (Guerreiro et al., 2013), and it will be interesting to assess whether the binding of soluble TREM2 has a functional effect on neurons or on plaque dynamics. A previous paper showed positive effects of sTREM2 in microglial proliferation (Zhong et al., 2017).

The combined findings of the paper strongly support a partial loss of function of the R47H variant in several aspects, including decreased binding to certain ligands and impaired activation of microglia. Thus, it will be interesting to further investigate how TREM2 variants could contribute to the pathogenesis of AD.